Tailoring and optimization of a honey-based nanoemulgel loaded with an itraconazole–thyme oil nanoemulsion for oral candidiasis

Abstract The use of essential oil–based nanoemulsions (NEs) has been the subject of extensive research on a variety of conditions affecting the oral cavity. NEs are delivery methods that improve the solubility and distribution of lipid medicines to the intended areas. Because of their antibacterial and antifungal properties, itraconazole and thyme oil–based self-nanoemulsifying drug delivery systems (ItZ-ThO-SNEDDS) were created to protect oral health against oral microorganisms. The ItZ-ThO-SNEDDS were created utilizing an extreme verices mixture design, and varying concentrations of ThO (10% and 25%), labrasol (40% and 70%), and transcutol (20% and 40%) were used. The ItZ-ThO-SNEDDS had droplet sizes of less than 250 nm, a drug-loading efficiency of up to 64%, and a fungal growth inhibition zone of up to 20 mm. The accepted design was used to obtain the ideal formulation, which contained ThO in the amount of 0.18 g/ml, labrasol 0.62 g/ml, and transcutol 0.2 g/ml. The best ItZ-ThO-SNEDDS formulation was incorporated into a honey-based gel, which demonstrated improved release of ItZ in vitro and improved transbuccal permeation ex vivo. In addition, when compared with various formulations tested in rats, the optimized loaded emulgel decreased the ulcer index. This study therefore demonstrated that the ItZ-ThO-SNEDDS could offer an effective defense against oral diseases caused by microbial infections.


Introduction
For overall health and a high quality of life, oral health is crucial. In 2012, the World Health Organization defined oral health as the absence of mouth or facial pain, oral infections or sores, and other disorders that restrict a person's ability to engage in with his daily and psychosocial activities (World Health Organization, 2012). Oral diseases continue to be a neglected area of global health despite the significant social and financial costs they impose on many nations (FDI World Dental Federation, 2015). Most adults have tooth decay, and 15% to 20% of middle-aged adults have serious gum disease (Buset et al., 2016). Poor dental health may be linked to such diseases as diabetes, heart disease, stroke, pneumonia, and other respiratory illnesses, according to the literature (Silva et al., 2015). Another typical reason for headaches and ear and facial pain is disorders of the jaw (Katz et al., 2010).
Dental clearance prior to medical procedures such as cardiac surgery, cancer/bisphosphonate therapy, and radiation therapy reduces the systemic and oral side effects of these procedures (Rajan et al., 2014).
Free-living eukaryotic organisms known as fungi can be molds (filamentous fungi), yeasts (round fungi), or a blend of both (dimorphic fungi) (Terezhalmy & Huber, 2011). One of the common fungal infections of the oral mucus membranes is oral candidiasis (Prasanna, 2012). It is caused by the yeast Candida albicans, which is a fairly normal component of the oral microbiota; it is found in 30% to 50% of people. As people age, the rate of carriage rises (Dangi et al., 2010). In 60% of patients over 60 years of age with dental problems, C. albicans is recovered from their mouths (Parihar, 2011). In addition to C. albicans, there are several other species of Candida that can be found in the oral environment, including Candida guilliermondii, Candida krusei, Candida glabrata, Candida parapsilosis, Candida pseudotropicalis, and Candida tropicalis (Dangi et al., 2010).
Itraconazole (ItZ) is a triazole antifungal that can be taken orally and has a propitious pharmacokinetic profile. It has been shown to have a wide range of efficacy (Chong & Sullivan, 2007). ItZ has been demonstrated in noncomparative clinical trials to be highly effective when delivered once or twice daily for a variety of superficial and more consequential 'deep' fungal ailments (DiMasi et al., 2003). This drug's original purpose was to act as a broad-spectrum antifungal drug by inhibiting 14-α-lanosterol demethylase (14LDM), an enzyme that causes ergosterol to be produced in fungi and cholesterol to be produced in humans (Okabayashi et al., 2009). It is used to prevent immunosuppressive diseases, as well as treat fungal infections such aspergillosis, candidiasis, and histoplasmosis (Chang et al., 2017). ItZ has uncommon side effects such as neutropenia, liver failure, and heart failure but is generally considered to be safe (Girois et al., 2006).
For centuries, people have used the thyme plant and its EOs to treat infections related to the upper respiratory tract, the symptoms of bronchitis, pruritus brought on by dermatitis, bruises, and sprains (Kohlert et al., 2002). These days, it is frequently used in dental medicine as a disinfectant and as an expectorant for coughs brought on by colds (Nilima et al., 2013). It has antibacterial effects on both Gram-positive and Gram-negative bacteria, as well as antifungal, antispasm, anti-inflammatory, antioxidant, and antiviral activities against human rhinoviruses, influenza viruses, and herpes simplex virus type I (Walther et al., 2020). There have been no cases of toxicity when the oil is taken at levels regularly used, and therefore it is generally regarded as safe (Eva et al., 2018;Salehi et al., 2018).
Several antimicrobial medicines have lost their efficacy for treating wounds owing to the development of treatment-resistant microbes (Guo & Dipietro, 2010). The use of honey for wounds is one of the alternative medicines that have been sought out more widely used in clinical settings for the treatment of wounds (Molan, 2001). Honey's inherent qualities and active ingredients are essential for the process of healing in wounds (Molan & Rhodes, 2015). Natural honey has a jelly-like viscosity and forms a superficial film covering wounds and lesions and prevents bacteria and dehydration (Langemo et al., 2009;Sell et al., 2012). Because of its high sugar content, honey produces a strong osmotic gradient that draws fluid up the subdermal tissue and provides a source of glucose for the thriving cellular components in wounds (Molan, 2002). Honey's water activity is lower than 0.91 aw, and this inhibits and limits bacterial development on injured sites and promotes fluid movement that causes debris and necrotic tissue to be sloughed from the wound (Gleiter et al., 2006;Sundoro et al., 2012).
A breakthrough idea called nanomedicine has shown great promise for delivering prospective medications with low solubility and bioavailability (Salem et al., 2022;Hussein et al., 2022;Alhakamy et al., 2022). A nanoemulsion (NE) is a delivery system that can improve solubility and stability . Widely researched as drug delivery platforms, NEs are made up of nanosized oil droplets stabilized by a combination of surfactants and co-surfactants . Using EO-based NEs to combat oral bacteria is a viable strategy . Gels are relatively new dosage forms often generated when significant quantities of aqueous, hydroalcoholic, or nonpolar vehicles are mixed in a three-dimensional (3D) network of polymeric components . However, they are less effective at delivering hydrophobic medications (Wong & Dodou, 2017). Emulgels, a hybrid of emulsions and gels, were therefore a viable alternative that would allow introducing hydrophobic medications topically (Talat et al., 2021). These dosage forms are well liked because they combine the benefits of both emulsions and gels, like regulated drug release of emulsions and excellent thermodynamic stability of gels (Laffleur & Keckeis, 2020).
In order to gather the most data with the fewest experiments, and provide an explanation for any experimental errors, statistical experimental designs were developed (Gregg Stetsko Manager, 1986). Utilizing these designs has the added benefit of requiring that formulations be made carefully and that statistical laws be followed. This means that scientists must be precise when setting up the experimental goals and the procedures for meeting those goals. Additionally, the design can forecast the behavior of the ideal formulation . Because statistical designs frequently lead to an optimum formulation, they can be a cost-effective method of study (Alkhalidi et al., 2018). In order to create a novel formulation for the successful treatment of oro-mucosal ulcers brought on by oral candidiasis, the current study targeted the development of a honey-based nanoemulgel containing an itraconazole and thyme oil-based nanoemulsion (ItZ-ThO-NE) and evaluated its antibacterial and antifungal efficacy and calculated its ulcer index.

Materials
The beauts-way Commercial Co., Ltd. (Jeddah, Saudi Arabia) provided the ItZ and ThO. The Saudi Arabian Japanese Pharmaceutical Company Ltd generously donated the PVP and PEG (SAJA, Jeddah, Saudi Arabia). Agar and Laureth-20 were acquired from the Sigma Water Import and Export Co., Ltd. (Shaanxi, China). Tween and Cremophor EL, as well as isopropyl alcohol and glycerin, were procured from Sigma Aldrich (St. Louis, Missouri, USA). Labrasol and transcutol were generously provided by Gattefosse (Saint-Priest, France), and the honey was acquired from the Hali Health Company (Ras Al Khaimah, UAE). The other compounds that were utilized in this experiment were all of analytical grade. Purified water was used in the experiments.

Solubility studies
To determine the variable self-emulsifying regions that may be used to construct ThO-based NEs containing ItZ, the drug solubility was evaluated in a variety of surfactants and co-surfactants .
To determine the drug's solubility in each specific solution, excess quantities of ItZ were dissolved in 3 ml of the surfactants, namely, Tween, labrasol, Laureth-20, and Cremophor EL, as well as the co-surfactants transcutol, isopropyl alcohol, glycerin, HCO-20, and propylene glycol. The combinations were kept in vials for 3 days at 25 ± 2 °C in a water bath. After the required balance was achieved, the mixtures were precipitated by centrifugation for 20 minutes at 4000 rpm to remove any extra medication. In order to determine the ItZ content of the supernatants, a high-performance liquid chromatography (HPLC) method using a C18 HS (250 × 4.6 mm) column was employed (Thimmaraju et al., 2012). Acetonitrile and double-distilled water were combined in this procedure's mobile phase in a ratio of 90:10 v/v at a flow rate of 1.0 ml/ min. A UV detector set at 263 nm was used for the detection. It was discovered that ItZ had a retention time of 7.5 minutes. ItZ also had a linearity range of 4 to 60 µg/ml and a linear regression coefficient of 0.991.

Itz-ThO emulsifying regions' tenacity in particular surfactants and co-surfactants
In a review to determine the lower and higher limits for every ingredient of the self-nanoemulsifying drug delivery systems (SNEDDS), which were chosen based on solubility, the self-emulsifying regions were located using pseudoternary phase diagrams. When diluted with water and gently stirred within those limits, blends of ItZ, ThO, the chosen surfactant (labrasol), and the chosen co-surfactant (transcutol) spontaneously created a pellucid emulsion with globules in the nanometer range. When the droplet size is greater than 1 nm but smaller than 1 m, the emulsion area in the phase area can be recognized as an NE region. The ItZ-loaded NEs were then tested.

Design of ItZ-ThO self-nanoemulsifying formulations and optimization of experiments
Because of its great effectiveness in evaluating the emulsions and projecting the best solutions, an extreme vertices mixture experimental design (Table 1) was used for the creation and selection of the best ItZ-ThO-SNEDDS. To ascertain the impacts of the ThO level (A), labrasol level (B), and transcutol level (C) in 17 formulations, a mixed design based on extreme vertices was used. The prepared SNEDDS' globule size (Y 1 ), drug-loading efficiency (Y 2 ), and growth inhibition of C. albicans (Y 3 ) were the dependent responses. The response factors were noted and optimized utilizing the Design of Experiments.

Visual screening of ItZ-ThO-SNEDDS
Visual inspection was used to assess the effectiveness of the production of the ItZ-ThO-SNEDDS in terms of their clarity and stability; this included defects such as coalescence or the shattering of spontaneously generated NEs .

Assessment of the nanoemulsions' droplet sizes
Double-distilled deionized water, 400 ml, was used to dilute 100 ml of each formulation, and the blend was mixed using a vortex mixer for 5 minutes. Aliquots of 200 µl were taken from the diluted samples to determine the droplet size and polydispersity index (PDI) using a Zetatrac and a dynamic light-scattering approach (Microtrac, Inc., Montgomeryville, Pennsylvania, USA) .

Analysis of ItZ-ThO-SNEDDS' drug-loading efficiency
By independently dispersing a known excess quantity of ItZ in 1 g of a plain SNEDDS, the ItZ loaded in each SNEDDS mixture was calculated. The mixtures were placed in vials and kept in a shaking water bath at 25 °C for 24 hours. The blends were precipitated by centrifugation for approximately 15 minutes at 4500 rpm after reaching equilibrium. The precipitates were gathered, thoroughly cleaned, and then dissolved in methanol. The amount of ItZ was calculated using the previously described HPLC procedure after the drug had been removed. The following mathematical formula was used to calculate the drug-loading capacity : Drugloadingefficiency c Product sdrug content mg Total product w weight mg u100 Eqn. (1)

Valuation of growth inhibition zone against Candida albicans
The disk diffusion method was used to assess the formulation's antifungal effectiveness. To prepare C. albicans strains for inoculation and conduct an in vitro antifungal evaluation utilizing the fungus strains, the strains were grown on Sabouraud dextrose agar medium   saline, and thoroughly mixed. The 0.6-meq McFarland standards were used to preserve the uniform turbidity of the microbial suspension. To obtain the grass culture, streaks were produced on the Sabouraud dextrose agar media by inserting a sterilized cotton swab into the prepared fungal suspension.

Disk diffusion technique:.
To prepare the test and positive control samples, sterile filter paper disks measuring 5 mm in diameter were submerged in 100 µl of the ItZ-ThO-SNEDDS and ethyl alcohol (99.9%), respectively. The paper disks were inserted into seeded Sabouraud dextrose agar media after being soaked. The infected plates were incubated for 48 hours at 24 ± 2 °C before they were examined for each zone of inhibition.

Optimization of ItZ-ThO-SNEDDS
For the analysis of variance (ANOVA) of the resulting models, the F-ratio, p-value, and degrees of freedom were computed for the investigated parameters and their interactions. The responses were utilized to choose the model that would be most suitable for the data based on the results. The p-values below .05 showed that the tested model predictors were significant. The model fitness was further evaluated using the coefficient of variation (CV%) values, determination correlations, anticipated R 2 value, and adapted R 2 value. In this way, the NEs with the smallest globule size and highest drug-loading efficacy were identified.

Choosing the optimized ItZ-ThO-SNEDDS
The development and evaluation of the formulations involved evaluating the drug-loading capability and globule size. The formulations were then examined for their ex-vivo permeation, in-vitro drug release, and ulcer index.

Preparation of honey-based nanoemulgel loaded with ItZ-ThO-NE
An optimized ItZ-ThO-SNEDDS-loaded honey nanoemulgel was created by combining 5% (v/v) honey with 10% (w/v) PVP, 1% (w/v) agar solution, and 2% (v/v) PEG. An aqueous PVP solution made by dispersing 10 g of PVP in 60 ml of distilled water was used to create the simple honey gel (i.e. formulation F1), which was allowed to sit at room temperature (25 °C) overnight. To create the agar blend, 1 g of agar was solubilized in 20 ml of distilled water, heated until clear, and stirred constantly before 2 ml of PEG was added. Fifteen ml of honey was introduced when the mixture's temperature dropped below 45 °C after the homogeneous mixture had been in a sonicator water bath for an hour at 37 °C. The same procedures were followed in the preparation of an optimized honey nanoemulgel (i.e. formulation F2) that was loaded with the optimized ItZ-ThO-SNEDDS, but 10 g of PVP was dissolved in 60 ml of the NE created by dissolving 10 ml of the ItZ-ThO-SNEDDS in 50 ml of distilled water and stirring for 5 minutes before adding PVP. The remaining ingredients were then added as in the previously prepared plain honey gel. Additionally, four different hydrogel formulations were prepared for comparison (formulations F3 to F6). ItZ as a powder and ThO as an oil were used to make the honey gel for F3; that is, a SNEDDS was not used. The nanoemulgel for F4 was created without adding honey. The F5 honey gel, manufactured in the same way as the F3, did not contain ThO, and the F6, prepared in the same way as the F4, did not contain ItZ powder. The produced gels were evaluated for the following characteristics.

In-vitro dissolution of the hydrogel formulations.
In this study, the USP Dissolution Tester (Apparatus I) was used. Instead of using baskets, glass cylindrical tubes measuring 2.7 cm in diameter and 10 cm in length were attached to the spinning shafts and securely sealed with semipermeable membranes of 100 µm in pore size. These tubes contained the formulations to be assessed. The tested formulations (F2 to F5) each weighed 10 g and contained 50 mg of ItZ. Each glass tube containing one of the formulations was submerged in 50 ml of phosphate buffered saline (PBS, pH 6.8). The medium was agitated at a speed of 25 rpm while the release study was conducted at 37 ± 0.5 °C.
Over 1 hour, samples were taken out of the dissolved mixture at various intervals. Using the previously described HPLC procedure, the absorbance was measured to determine how much ItZ had been released .

Ex-vivo transmucosal penetration test of the hydrogel formulations. Ex-vivo tests were conducted on
the ItZ aqueous suspension and various manufactured formulations (F2 to F5), each of which contained 5 mg/ml of ItZ. An automated Franz diffusion cell (MicroettePlus, Scilogex LLC, Rocky Hill, Connecticut, USA) was used as the apparatus, and sheep buccal mucosal membrane obtained from a nearby local butcher was used as the model penetration membrane. The Franz diffusion cell's donor and receptor compartments were adequately separated by the processed sheep buccal mucosa. The exposed surface area of the receptor compartment covered by the membrane was 1.75 cm 2 . The medium was agitated at a rate of 400 to 450 rpm while being kept at a temperature of 37 ± 0.5 °C in the drawing chamber, which encompassed 8 ml of PBS (pH 6.8).
At regular intervals, precise aliquots were automatically removed, and the previously described HPLC method was used to quantify the ItZ content. A graph was made to show how long it took for the ItZ to penetrate the Q per cm 2 of the membrane; this finding let researchers better understand how the drug was dispersed throughout the mucosa. From the acquired diffusion data, significant parameters such as the Jss (steady-state flow), Pc (permeability coefficient), EF (enhancement factor), and D (diffusion coefficient) were determined. Plots were made of the comparative dissemination patterns for various formulations. The following equation (2) was used to determine the percentage of ItZ that penetrated the membrane and the overall amount of ItZ distributed throughout the receptor chamber : Eqn. (2) where LT was the initial concentration of ItZ in the donor chamber and Lp was the quantity of ItZ that was transferred into the receptor compartment.

Assessment of the growth inhibition zone against
Candida albicans. By using the previously indicated disk diffusion technique, the antifungal activity of the investigated formulations (F1 to F6), as well as the aqueous dispersion of ItZ, was assessed.

Ulcer index evaluation.
Male albino rats weighing 150 to 250 g were used for this investigation. The in-vivo study was performed according to the institutional guidelines of the Animal Ethics Committee of Cairo Agriculture for Experimental Animals,Cairo,Egypt,. The ulcer index was assessed for seven animal groups, each consisting of three animals, using a previously described methodology (Alkhalidi et al., 2020). Group 2 received the ItZ aqueous suspension and was the positive control. Group 3 received formulation F1; Group 4 received the optimized nanoemulgel, or formula F2; Group 5 received formulation F4; Group 6 received formulation F5; and Group 7 received formulation F6. Group 1 received only normal saline and was the negative control.
After 3 days of therapy with each formulation, the ulcerative effect on the oral mucous membranes of every treated group was graded as 1 (normal-colored epithelial lining), 2 (red-colored epithelial lining), 3 (ulcers < 1 mm), 4 (ulcers > 1 mm but with no bleeding areas), and 5 (ulcers > 1 mm and with hemorrhagic streaks).

Assessment of ItZ solubility
The ItZ had the best level of solubility when it was mixed with the surfactant labrasol and co-surfactant transcutol, according to the investigations into its solubility in various surfactants and co-surfactants. Figures 1 and 2 show the relationship between the types of surfactants and co-surfactants utilized and the percentage of ItZ solubilized. In order to determine the highest and lowest limits of each component of the SNEDDS, labrasol and transcutol were chosen for the pseudoternar y phase diagram construction.

The ability of certain surfactants and co-surfactants to emulsify ItZ-ThO-EOs with a high degree of tenacity
In order to stabilize the system created by combining water and oil, a surfactant is frequently employed, with the goal of reducing the particle size. According to Tsai et al. (2014), the oil phase typically consists of natural triglycerides, hydrocarbons, and other nonpolar hydrophobic molecules, while the aqueous phase typically contains solutes and electrolytes that have been dissolved in water. To accurately pinpoint the range of concentrations of the thyme EO, labrasol, and transcutol that should produce regions of NE, a pseudoternary phase diagram was built for the current study.
The precise concentration levels of thyme EO, labrasol, and transcutol for making NEs were found to be 10% to 25%, 40% to 70%, and 20% to 40%, respectively, as can be seen in the pseudoternary phase diagram in Figure 3. These concentration ranges were used to create the statistical design that was later used for the creation and optimization of several ItZ-ThO-SNEDDS formulations.

Visual examination of ItZ-ThO-SNEDDS
An examination of the ItZ-ThO-SNEDDS with the naked eye revealed translucid dispersions that were clear and did not exhibit any coalescence or breakage. This implied that the concentration ranges that were specified were exact in the establishment of stable NEs.

Assessment of ItZ-ThO-SNEDDS' droplet size
The NEs had droplet sizes of 84 ± 2.1 to 240 ± 3.0 nm (Table 2) and PDI values between 0.09 and 0.32. This demonstrated the formulations' uniformity, stability, and size distribution.
The collected globule size data were subjected to a special quadratic model of polynomial analysis. The researched model was effective at determining the substantial impact of the thyme EO (A), labrasol (B), and transcutol (C) on the ItZ-ThO-SNEDDS' droplet sizes, according to the chosen mathematical design. As shown in Table 3, the model had a modulated R 2 value of 0.9991 and a predicted R 2 value of 0.9960, which were closely correlated. An ANOVA data analysis produced the equation shown below: Droplet The perturbation, 3D surface, and contour plots in Figure 4 show how the parameters affected the size of the ItZ-ThO-SNEDDS droplets. The graphs demonstrate how the amount of different formulation variables in the formulations affected the globule size of the generated NEs.
According to the graph, labrasol (i.e. factor B) had the biggest impact on the droplets' size. The increase in surfactant limits reduced the size of the globules, as shown in Figure 4(A), since this caused a lowering of the interfacial tension that existed between the aqueous and nonaqueous phases . The fact that increasing the percentage of oil reduced the surfactant and co-surfactant levels and, therefore, limited their capacity to minimize the droplet size may help to explain the increase in droplet size in relation to the increased amount of ThO. Larger oil globules were produced as a result, and this was consistent with the outcomes mentioned in the literature (Ziani et al., 2011;Pavoni et al., 2020). Moreover, boosting the ThO level might have allowed for more ItZ, which is a lipophilic agent, to be incorporated into the oily droplets of the NE and, hence, for bigger droplets to be attained.

Assessment of ItZ incorporation efficiency within ItZ-ThO-SNEDDS
As demonstrated in Table 2, the loading of ItZ in the NEs ranged between 30 ± 1.5% and 64 ± 1.1%.
The resulting drug-loading data were subjected to a special quartic model of polynomial analysis. The ItZ-ThO-SNEDDS' drug-loading capacity was evaluated using the model to assess the major impacts of ThO (A), labrasol (B), and transcutol (C). As shown in Table 3 Figure 5 shows the main effect, contour, and 3D surface plots that illustrate how the investigated parameters affected the drug loading in the ItZ-ThO-SNEDDS.
As might be noticed from the preceding plots, the ThO level (i.e. factor A) significantly increased the incorporation of ItZ into the NE's droplets. This finding might be due to the great capacity of the oil to absorb ItZ because of the lipophilic nature of ItZ. Further, improving the percentage of oil in the emulsion would lower the accompanying levels of labrasol and transcutol and, hence, lessen their ability to force the drug into the surrounding aqueous milieu . Additionally, as can be seen from the graph, large amounts of labrasol or transcutol were associated with a lower loading capacity of ItZ in the NE. These outcomes could be ascribed to the comparable decrease in oil levels and, hence, the decreased space into which ItZ could be incorporated in the NE droplets. It could also be related to the interfacial tensionlowering properties of labrasol and transcutol.

Evaluation of fungal growth inhibition zones of ItZ-ThO-SNEDDS against Candida albicans
As is shown in Table 2, the inhibition zones against C. albicans created by the NE formulations ranged from 5 ± 0.5 to 20 ± 1.0 cm. The zone inhibition data were fitted to a special quartic model of polynomial analysis. The ItZ-ThO-SNEDDS' growth zone inhibition capabilities were examined using the selected statistical model to assess the major impacts of ThO (A), labrasol (B), and transcutol (C). As shown in Table 3 Figure 6 shows the main effect, contour, and 3D surface graphs that illustrate how the investigated parameters impacted the fungal growth inhibition zones of ItZ-ThO-SNEDDS against C. albicans.
As might be seen from the above graphs, ThO has a great capacity for expanding the growth inhibition zones of C. albicans. Ergosterol is a special sterol that can only exist in the outer membrane of a fungus and is crucial to its healthy development and operation. As a result, substances affecting the level of ergosterol might have antifungal properties (Kowalczyk et al., 2020). Thymol, the major component of ThO, has potential antifungal action that is based on how it affects the metabolism of fatty acids, especially ergosterol, in fungal cells (De Lira Mota et al., 2012). Among other things, it produces oxidative stress and an increase in reactive oxygen species, and this lowers the levels of capsular polysaccharide and the extracellular polymer matrix (EPS) (Al-Shahrani et al., 2017). In cell membranes of Candida and Cryptococcus that had been exposed to thymol, ergosterol  levels decreased, as previously stated in the literature. This resulted in disruptions of the membrane integrity and membrane-associated enzymes, significant damage, and, ultimately, cell death (Poonam et al., 2019).
The decrease in the size of the inhibition zones associated with the increase in labrasol (B) and transcutol (C) levels might be correlated with the comparable decrease in the level of ThO and, hence, its lowered ability to destroy the fungal cell membrane. The decrease in the area of the growth zone area observed when the surfactants were increased might be explained by the expected decrease in ItZ loaded into the NE and, hence, the decreased overall antifungal activities of the formulations.

Itz-ThO-SNEDDS optimization
The optimized NE formulation was created using the most appropriate qualities revealed by the aforementioned data. The best combination was composed of 0.18 g/ml of ThO, 0.62 g/ml of labrasol, and 0.2 g/ml of transcutol, according to Statgraphics software, which also suggested many other options that represented different combinations of the variables under study. The optimal formulation had a drug-loading capability of 67%, a droplet size of 105 nm, and an inhibition zone of 18.177 mm, and it acquired a desirability of 0.931. The desirability ramp is shown in Figure 7, which also shows the levels of the independent variables and the anticipated values of the measured responses for the best formulation. Table 4 shows that the parameters of the optimum formulation's veritable and anticipated values were very nearly identical, with no appreciable discrepancies (p > .05), and this supported the accuracy, validity, and precision of the equations.

In-vitro dissolution behavior of the ItZ-ThO-SNEDDS-loaded hydrogel formulations
The evaluated formulations' drug release profiles are shown in Figure 8. The drug suspension had a very low cumulative  percentage of the drug released (22 + 2%) after 60 minutes; this might be due to the low water solubility of ItZ. Formulation F4, which was a nanoemulgel without honey, had a drug release percentage of 85 ± 3%. Such a high percentage of release of the optimized formulation compared with the other examined formulations might be attributable to the NE content of amphiphilic molecules, which may improve drug release by enhancing ItZ dissolution . Additionally, the small globule size of the NEs provided a broader surface area for drug release and aided in increasing the amount of drug released, enabling a higher amount of absorption and a quicker initiation of the drug's effect . In addition, the lack of honey in the formulation might have contributed to a decrease in the formulation's viscosity, which could be expected to enhance the ItZ release. Meanwhile, formulation F2, which contained the optimal nanoemulgel and honey, had a drug release percentage of 72 ± 4%. The decrease in ItZ release from formulation F2 compared with formulation F4 could be due to the higher viscosity of the F2 with its honey content, which might have restrained the drug release (Salem et al., 2022). Formulation F3, which contained ItZ powder and ThO dispersed in a gel base, attained a percentage of release of 39 ± 4% of ItZ. Such a low release percentage may have resulted from poor ItZ solubility due to the lack of amphiphilic molecules in this formulation compared with the gel contained in the NE of same components (i.e. F4 and F2). Formulation F5, which contained ItZ powder in a gel base but no ThO, had a percentage of release of 19 ± 1% of ItZ. This low result might have been due to the presence of ItZ in a powder form but no surfactants that could have solubilized it, as well as to its viscous gel base, which further hindered its diffusion and release.

Ex-vivo transmucosal penetration test of the hydrogel formulations
As might be observed from Table 5, formulation F2, which was composed of the optimal formulation-loaded hydrogel that contained honey, achieved a Q 24 value of 3125 μg/cm 2 with a 4.16-fold increase in permeation compared with the ItZ aqueous suspension, which had a Q 24 of 750 μg/cm 2 . Such a result demonstrates that the NEs were significantly involved in enhancing the ItZ penetration through the mucosa. The NEs' vital role in increasing drug permeation can be ascribed to its content of surfactant, which might contribute to drug diffusion from the formulation and exert a considerable effect on fluidizing the mucosal cell membrane, leading to enhanced ItZ permeation (Salem et al., 2020). In addition, the small size of the NE globules provided a larger surface area for drug permeation. Simultaneously, formulation F4 had a Q 24 of 2614 μg/cm 2 . Such a decrease in the Q 24 compared with F4,   even though both contained the ItZ NE, might be due to the presence of honey in F2. This could provide greater viscosity, which might have allowed for more intimate contact and a prolonged residence time of the formulation within the mucosa and, thus, a greater chance for ItZ to permeate the mucosal membrane (Laffleur, 2014). Moreover, F3, which contained ItZ powder and ThO dispersed in a gel base, had a Q 24 of 1882 μg/ cm 2 , while F5, which contained ItZ powder in a gel base but no ThO, had a Q 24 of 800 μg/cm 2 . Such a large difference in the Q 24 values of these two formulations may be attributable to the oil's potential to increase permeability by altering and modifying cell membrane characteristics, such as by boosting ItZ solubility and penetration through the mucosa or by improving the membrane fluidity, as was previously described in the literature Jiang et al., 2017).

Ulcer index determination
The data in Table 6 show that F2 (i.e. the optimum ItZ-ThO-SNEDDS-loaded gel) had the lowest ulcer index. It was comparable to that of the negative control group, which received normal saline, and Group 3, which received plain gel. This finding revealed the substantially low ulcer-forming capacity of F2. Such noticeable activity might be ascribed to the nature of the gel base carrying F2, which had honey among its components. Honey has certain physical traits that encourage its application as a medicinal drug to fight certain infections (Hixon et al., 2019). These characteristics of honey are also connected to its ability to reduce inflammation and promote wound healing, antioxidant and the capacity to scavenge free radicals. It is an immune stimulator that can improve the immunological response (Almasaudi et al., 2016). It can be used to treat a variety of ulcers; it can also be utilized for wound healing and skin sanitation. In addition to its anti-inflammatory and bacteria-fighting capabilities, it accelerates the healing of wounds and reduces the ulcer index (Almasaudi et al., 2017). These results were further confirmed when they were compared with the results for Group 2, which received the aqueous dispersion containing the drug and had an ulcer index of 5. Group 2 received ItZ twice daily for 14 days, and although ItZ has antifungal properties, because it is a synthetic drug it contributed to the injury of the buccal mucosal membrane. Groups 5 and 6, which received F4 and F5, respectively, both had ulcer indexes of 3. The change in the ulcer index for Groups 5 and 6 from that of Groups 3 and 4 might be due to the lack of honey in Group 5 (which received F4) or the lack of ThO in Group 6 (which received the gel containing ItZ powder only). Such outcomes emphasize the ulcer-reducing properties of both honey and ThO. The ulcer-healing activity of ThO was further proved by the results for Group 7, which received F6, which was composed of the gel loaded with NE but without ItZ; this group had an ulcer index of 2. This low ulcer index may have been due to the ThO content. ThO is well known for its anti-inflammatory and antioxidant properties, which contribute to its ability to heal lesions and ulcers; this is due to its main phenolic compounds, thymol and carvacrol (Nagoor Meeran et al., 2017;Kohlert et al., 2002).

Conclusion
ItZ was effectively developed as a functional NE in combination with ThO. A pseudoternary phase diagram was used to estimate the ideal concentrations of ThO, labrasol, and transcutol in combination to create the necessary drug delivery system. The created systems appeared to be reasonably stable, as evidenced by the NEs, which had globule sizes between 84 ± 2.1 and 240 ± 3.0 nm with acceptable homogeneous distribution. Additionally, the created formulations had a drug-loading efficiency of between 30 ± 1.5% and 64 ± 1.1% and a fungal growth inhibition zone of up to 20 mm. The extreme vertices mixture design was used to obtain the best formulation, which consisted of the variables ThO, labrasol, and transcutol at levels of 0.18 g/ml, 0.62 g/ ml, and 0.2 g/ml respectively. The optimal formulation was loaded into a honey-based oral gel and showed enhanced release and transmucosal permeation. When tested in rats, the optimized ItZ-ThO-SNEDDS-loaded gel displayed the lowest ulcer index value when compared with other formulations. Overall, this study showed that ItZ-ThO-based NEs in combination with a honey-based gel could offer an effective defense against oral diseases brought on by microbial infections.